Photosensitizer and method for production thereof

ABSTRACT

The invention relates to medicine and concerns a photosensitizer for detecting and curing tumors. The inventive photosensitizer is embodied in the form of a composition containing chlorin in the form of salt and alkali metals. The chlorin is composed of 80-90% of chlorin e 6 , 5-20 of purpurin 5 and the rest being purpurin 18-chlorin p 6 . Said photosensitizer is produced by extracting  Spirulina  biomass with the aid of acetone. Afterwards said biomass is exposed to acid treatment, neutralization, hydrolysis, extraction of pheophorbide a, dissolution in acetone, addition of a strong base, neutralization and reprecipitation of chlorin e 6 .

The invention concerns the sphere of medicine, particularly the sphereof photodynamic therapy (PDT) with the use of biologically activecompounds.

Photosensitizers (PS) are used as therapeutic agents at PDT and asfluorescent labels at photodynamic diagnostics (PDD).

Mono-L-aspartil chlorin e₆ tetrasodium salt “Npe6” is known as PS (U.S.Pat. No. 4,977,177):

This PS is active at PDT.

Its disadvantages are: the laboriousness of producing, too high-speedtumour accumulation and excretion dynamics, that reduces the time ofeffective exposure on the tumour, and comparatively low extent ofaccumulation in malignant formation (tumour) because of high hydrophily,that activates only one of several possible mechanisms of tumourdestruction in the PDT process, namely only the blood vessel affection.

Lysil chlorin p₆ trisodium salt “LCP” is known as PS (U.S. Pat. No.5,330,741):

This PS is active at PDT.

Its disadvantages are: the laboriousness of producing and the fact thatit is a mixture of two monoamides at 13 and 15 positions in the ratio of10:1, that may lead to ambiguous biodistribution and excretion.

Pheophorbid a sodium salt is known as PS (U.S. Pat. No. 5,378,835):

This PS can selectively accumulate in malignant tumours and is active atPDT.

Its disadvantage is the tendency to oxidation (chemical instability) atstorage as a solution, not full solubility in water after storage as asolid, hydrophoby and, as a consequence, slow excretion out of theorganism, that leads to prolonged photosensitivity of skin integument.

Chlorin e₆ derivatives are also known as PS (U.S. Pat. No. 5,002,962):

-   -   wherein R=hydrophobic hydrocarbon substituent, saturated or        unsaturated, straight or branched, consisting of 4 to 25 carbon        atoms.

PS, wherein R is hexil, is tropic to malignant tumours and is aneffective agent for PDT.

Its disadvantages are: the laboriousness of preparative producing andpurification, high hydrophoby and, as a consequence, slow accumulationin tumour and low stability of water solutions of medicinal forms atstorage.

There known a method of producing PS, namely, composition of chlorins assalts with alkaline metals, destined for medical practice. This methodconsists of the following: plant (floral) biomass are extracted with a2:1 to 8:1 mixture of hydrocarbon consisting of 6-12 carbon atoms andalcohol consisting of 2-10 carbon atoms, resulting chlorophyll solutionis evaporated at the atmospheric pressure, alcohol consisting of lesscarbon atoms than extraction alcohol is added, hydrocarbon is fullydistilled off the mixture at the atmospheric pressure, alcohol alkalinesolution is slowly (gradually) added to the alcohol chlorophyllssolution at the alcohol boiling temperature, but less than 120° C., tillpH is 11.5-11.8, the mixture is cooled, incubated for 4 hours,filtrated, extracted with hydrocarbon consisting of 6-12 carbon atoms,alcohol phase containing magnesium complexes of chlorins is separated,alcohol is evaporated at the atmospheric pressure, hydrochloric acid isadded to the residue till pH is 3.5, the mixture is incubated till theend of chlorin precipitation and filtrated, the precipitate is dissolvedin methanol, alcohol alkaline solution is added till pH is 8.5, the PSsolution is filtrated and evaporated in vacuum (U.S. Pat. No.3,102,891).

The disadvantages of this method are: the use of high temperatures whileremoving solvents out of extract, the use of alcohols, especially methylalcohol, that leads to the allomerisation of E exocycle and formation ofmultiple different oxidation products of pheophytins and pheophorbids(K. Hyvarinen, J. Helaja, P. Kurchen, I. Kipelainen, P. H. Hynninen. H-1and C-13 NMR spectra of the methanolic allomerization products of13(2)-(R)-chlorophyll Magnetic Resonance in Chemistry.-1996.-V.33.-N8.-p. 646-656), this leading to the complex mixture the compositionof which is undefined and hard to reproduce.

There known a method of producing PS, namely, chlorin e₆ sodium salt,consisting of the following: 1N NaOH solution is added to the chlorin e₆trimethyl ether solution in tetrahydrofuran, the mixture is stirred for2 days at the room temperature under nitrogen, water is added to themixture, then organic solvent is extracted with methylene chloride, thetraces of the latter are eliminated by bubbling nitrogen through thechlorin e₆ salt solution (U.S. Pat. No. 5,002,962).

The disadvantages of this method are: low availability of sufficientamounts of starting chlorin e₆ trimethyl ether, long duration ofproducing the PS due to chemical inertness of ester radical at the13^(th) position of tetrapyrrol macrocycle and instability of PSmedicinal forms at storage in the form of water solution due toincomplete saponification of ester group at the 13^(th) position of themacrocycle.

There known a method of producing PS, namely, “LCP” photosensitizer forphotodynamic therapy (trisodium salt of lysyl-chlorin p₆), consisting ofthe following: biomass is treated with acetone 2-3 times in order toextract chlorophyll a, the biomass is filtrated or centrifuged, theextract is evaporated, treated with acid in order to remove magnesiumion out of the chlorofyll molecule and to hydrolyse phytyl ester group,methyl alcohol being added for concurrent esterification, the reactionmass is treated with water, pheophorbid a derivative is extracted withchlorous methylene, the extract is neutralised, washed with water,evaporated, chromatographed on aluminium oxide, methylpheophorbid a iscrystallised out of the mixture of chlorous methylene-methanol and theresulting pheophorbid a derivative is reacted with strong inorganic basein the presence of oxygen in pyridine-diethyl ether-n-propanol, thereaction mass is treated with water, the water phase is acidified tillpH 4, “unstable chlorin” is extracted with chlorous methylene, theextract is evaporated, “unstable chlorin” is redissolved intetrahydrofurane, the solution is evaporated, this procedure is repeatedtill absorption at 700 nm stops to increase, the resulting purpurine 18is dissolved in tetrahydrofurane, esterified with diazomethane, thepurpurine 18 methyl ester is mixed with lysine water solution inchlorous methylene in the presence of pyridine, the mixture is stirredfor 12 hours at room temperature, the solvents are removed in highvacuum, then the resulting crude product is purified by reverse phasehigh-performance liquid chromatography (HPLC), the solvents are removedby lyophilisation, the PS is dissolved in phosphate buffer in order toobtain injection solution for PDT, 0.1N NaOH solution is added, pH isadjusted to physiological value of pH 7.35 with 0.1N HCl and thesolution is filtrated through microporous filter (U.S. Pat. No.5,330,741).

The disadvantages of this method are: low reproducibility,labouriousness (the use of high vacuum, crystallization, columnchromatography and HPLC, long duration of the reaction with lysine), theuse of high toxic and inflammable reagents (diazomethane, pyridine,methanol, tetrahydrofurane, diethyl ether). These disadvantages make themethod unsuitable for pharmacetical industry. Besides, the resultingwater soluble target product is stable only for 24 hours at 4° C. in thedark in the form of water solution, and in the form of solid substanceit is stable only for 4 months at 4° C. in the dark, while according tothe pharmacopoeia requirements it should be stable not less than for 6months (Leach M. W., Higgins R. J., Boggan J. E., Lee S.-J., Autry S.,Smith K. M. Effectiveness of a Lysylchlorin p₆/Chlorin p₆ mixture inPhotodynamic Therapy of the Subcutaneous 9L Glioma in the Rat. CancerResearch 1992, V. 52, 1235-1239). Moreover, as for the chemicalcomposition, this PS represents the mixture of monoamides at the 13^(th)and 15^(th) positions in the approximate ratio of 10:1 that may lead toits ambiguous biological distribution and excretion out of the organism.

The aim of this invention is to obtain PS that is characterised by easypreparative isolation go and purification, balancedhydrophoby-hydrophily and, as a consequence, by the optimal speed oftumour accumulation and excretion out of the tumour and the wholeorganism, and also by medicinal forms water solutions high stability atstorage.

This aim is achieved by producing PS comprising chlorin in the form ofsalt with alkali metal, chlorin being composed of chlorin e₆(13-carboxy-17-[2-carboxyethyl]-15-carboxymethyl-17,18-trans-dihydro-3-vinyl-8-ethyl-2,7,12,18-tetramethylporphyrin)

making up 80-90%, purpurine 5(13-carboxy-17-[2-carboxyethyl]-15-formyl-17,18-trans-dihydro-3-vinyl-8-ethyl-2,7,12,18-tetramethylporphyrin)

making up 5-20%, and purpurine 18-chlorin p₆(13-carboxy-17-[2-carboxyethyl]-15-carboxy-17,18-trans-dihydro-3-vinyl-8-ethyl-2,7,12,18-tetramethylporphyrin)

making up the rest, so that mentioned components form the composition,sodium and potassium may be used as alkali metal.

Also the aim of this invention is to achieve high reproducibility of themethod of producing PS, its simplicity, chemical stability of the PSmedicinal forms not less than for 1 year, as well as to achieve totalityof physicochemical and biological properties of PS that provide theeffectiveness of the PS at PDT, and also to avoid the use of toxicreagents.

The essence of the suggested method of producing PS is following:Spirulina biomass is treated with acetone till chlorophyll a iscompletely extracted, the biomass is filtered out or centrifuged, theextract is treated with acid in order to remove magnesium ion out of thechlorofyll molecule, the extract is neutralised and precipitatedpheophytin a is filtered out, then pheophytin a is hydrolysed in themixture of hydrochloric acid-acetone-hexane, 6-16 ml acetone, 0.6-6-mlhexane and 5-10 ml concentrated hydrochloric acid being used for every 1g of crude pheophytin a, the mixture is heated up to 40-60° C. andstirred for 20 min—1 hour, then hexane (6-16 ml) is added and organicphase is washed with the mixture of acetone and hydrochloric acid(2-10:1), water phase is washed with hexane, then water phase containingpheophorbid a is neutralised with excess of sodium citrate (tri-, di- ormono-substituted) water solution, precipitated pheophorbid a is filteredout, washed with water, recrystallised out of the acetone-water mixture,air dried till its weight becomes constant, then pheophorbid a isdissolved in acetone, strong inorganic base is added in the form ofwater solution of 0.05-1.00% concentration, stirred at 30-60° C. for5-30 min, extra volume of strong inorganic base is added in the form ofwater solution of 1-50% concentration, the mixture is heated at 40-60°C. for 20-90 min, neutralised with diluted hydrochloric acid, chlorin e₆precipitate is separated by centrifugation, washed with distilled watertill acid reaction disappears, 55-80% of chlorin e₆ is obtained, thenchlorin e₆ is recrystallised out of acetone in order to separate lineartetrapyrrols, chlorin e₆ is filtered out and washed with distilledwater, chlorin e₆ is heated in sealed reservoir at the temperatures of40-100° C. for 1 hour—30 days, then it is cooled and strong basesolution is added till pH 7.5-8.5, then the solution is adjusted withapyrogenic water for injections to make photosensitizer concentration6.5-7.5% _(mass).

Furthermore, in the method of producing PS after the stage of additionof strong base till pH 7.5-8.5 the mixture may be gel filtrated to makechlorin e₆ percentage up to 80-90%, purpurine 5—up to 5-20% andpurpurine 18—the rest, then diluted hydrochloric acid solution is addedtill photosensitizer precipitates, the solution is adjusted withapyrogenic water for injections to make photosensitizer concentration6.5-7.5% _(mass), so the “Liquid extract of chlorins” is obtained.

Moreover, in the method of producing PS after the stage of gelfiltration diluted hydrochloric acid solution may be added to thephotosensitizer solution till photosensitizer precipitates, then theprecipitate is filtered out or separated by centrifugation, theadditives approved by RF State Pharmacopeia are added till pH 7.5-8.5,apyrogenic water for injections is added to make photosensitizerconcentration 0.1-1% _(mass), then bacteria are filtered out.

Also in the method of producing PS after the stage of gel filtrationdiluted hydrochloric acid solution may be added to the mixture tillphotosensitizer precipitates, this precipitate is filtered out orseparated by centrifugation, adjusted with apyrogenic water forinjections to make photosensitizer concentration 6.5-7.5% _(mass), the“Liquid extract of chlorins” is dispersed in gel substrate according tohe following ratio: 0.5-12% _(mass) of the “Liquid extract of chlorins”,5-20% _(mass) of dimethylsulfoxide, the rest is water, the additivesapproved by RF State Pharmacopeia and gel substrate.

Furthermore, in the method of producing PS after the stage of gelfiltration diluted hydrochloric acid solution may be added to themixture till photosensitizer precipitates, this precipitate is filteredout or separated by centrifugation, adjusted with apyrogenic water forinjections to make photosensitizer concentration 6.5-7.5% _(mass), andthe resulting “Liquid extract of chlorins” is dissolved indimethylsulfoxide according to the following ratio: 0.5-12% _(mass) ofthe “Liquid extract of chlorins” and the rest is dimethylsulfoxide.

This method is realised with the use of standard laboratory chemicalpilot equipment: biomass is treated in 10-50 L aluminium vesselsequipped with mechanical stirrer, biomass is filtered through 5-20 Lnutch filters with oil vacuum pump and liquid nitrogen-cooled trap,biomass is centrifuged in the cooled floor centrifuge with 4×1 L bucketsand rotation speed of 6000 rpm, extract is acidified in glass 20 Lbottles, precipitated pheophytin a is filtered through 5-10 L nutchfilters with oil vacuum pump and liquid nitrogen-cooled trap, pheophytina is hydrolysed in 0.1-0.5 L heated three-neck round-bottom flasksequipped with stirrer, backflow condenser and feeding hole with stopper,solutions are washed in 2 L separating funnels, neutralised in 2-5 Lchemical beakers, pheophorbid a is filtered through 2-5 L nutch filterswith oil vacuum pump and liquid nitrogen-cooled trap, recrystallised in0.25-1 L chemical flat-bottom flasks, pheophorbid a is dissolved inacetone and reacted with strong inorganic base in 0.5-2 L heatedthree-neck round-bottom flasks equipped with stirrer, backflow condenserand feeding hole with stopper, chlorin e₆ precipitate is separated inthe cooled floor centrifuge with 4×0.5 L buckets and rotation speed of6000 rpm, chlorin e₆ is recrystallised in 0.25-0.5 L, 2-5 L chemicalflat-bottom flasks, chlorin e₆ is filtered through 1-2 L nutch filterswith oil vacuum pump and liquid nitrogen-cooled trap, chlorin e₆ isheated in 0.05-0.1 L round-bottom chemical flasks of heat-resistantglass, it is reacted with strong base solution and adjusted in 0.1-1 Lchemical beakers with the use of standard pH-meter andspectrophotometer, mixture is gel filtrated on a column of 50-10 mmdiameter and 100-150 mm height, bacteria are filtered out throughstandard 0.22 μm microporous filter of Millipore type, the “Liquidextract of chlorins” is dispersed in gel substrate with the use ofcutter or bead homogenizer, moreover, 0.01-10 L cone flasks withstoppers, 0.005-2 L cylinders, 0.05-2 L beakers, 20 L bottles, weigherwith 1-1000 g range and magnetic stirrers are used for preparing samplesand solutions; 5 L round-bottom flasks with thermometer and direct-flowwater condenser are used for acetone and hexane regeneration; rotaryvacuum evaporator is used for quick removing of solvents at lowtemperature.

According to the method of producing PS the concentrated hydrochloricacid solution is considered as saturated hydrogen chloride watersolution at the temperature of 20° C. that commonly contains 36-37%_(mass) of hydrogen chloride.

At the stage of pheophytin a turning into pheophorbid a the range ofhexane and acetone volumes (6-16 ml of acetone and 0.6-6 ml of hexane)is explained by the fact that if lesser volumes of solvents are usedpheophytin a dissolving is not complete and if the volumes are greaterthe solution would not be enough concentrated for fast hydrolysis. Therange of hydrochloric acid volumes (5-10 ml) is explained by the factthat if the volume is lesser the pheophorbid a yield decreases and ifthe volume is greater the reaction selectivity decreases due toformation of by-product pyropheophorbid a. The temperature range of40-60° C. is explained y the fact that if the temperature is lower thepheophorbid a yield decreases and if the temperature is higher thereaction selectivity decreases due to formation of by-productpyropheophorbid a. The time range of 20 nm-1 hour is explained by thefact that if this period is shorter the pheophorbid a yield decreasesand if this period is longer the reaction selectivity decreases due toformation of by-product pyropheophorbid a. The volume of hexane added(6-16 ml) is explained by the fact that if this volume is lesser theseparation of one of the reaction products, phytol, from the reactionmixture is not effective, and the use of greater hexane volume is notrational.

At the stage of pheophorbid a purification organic phase is washed withthe mixture of acetone and concentrated hydrochloric acid in the ratioof 2:1 to 10:1. If this ratio is lesser than 2:1 the flaky admixtureprecipitate is formed that is hard to separate from the water phasecontaining the target pheophorbid a. If this ratio is greater than 10:1the water phase becomes oversaturated with acetone and the admixturescome into it from the hexane phase, contaminating the target pheophorbida.

At the stage of pheophytin a turning into chlorin e₆ the strong baseconcentration lays in the range of 0.05-1.00%, its lower limit being theminimum that is necessary for the reaction of pheophorbid acyclopentanone ring (E ring) opening, and if he base concentration isgreater than 1% the E ring allomerisation (oxidation) reaction takesplace leading to “unstable chlorin” instead of the target chlorin e₆,and then leading to purpurin 18, and further—to chlorin p₆.

Then, according to the method, an extra volume of strong inorganic baseis added in the form of water solution of 1-50% concentration. If thisconcentration is less than 1% incomplete saponification of ester groupat the 13^(th) or 15^(th) position takes place. If the baseconcentration is greater than 50% tetrapyrrol macrocycle of PS opens insome cases.

Then the reaction mass is stirred at 30-60° C. for 5-30 min, the lessertemperature facilitating the E ring allomerisation process, and thehigher temperature facilitating chlorin e₆ decomposing to chlorin e₄.When adding an extra amount of strong inorganic base the temperaturerange is 40-60° C. and the time range is 20-90 min. If time andtemperature are less than stated the methyl ester at the 15² positionhas no time to hydrolyze, if time and temperature are greater by-productchlorin e₄ yield increases.

When chlorin e₆ turning into the “Liquid extract of chlorins” theprocess of oxidation and the subsequent thermolytic processes ofdehydration and decarboxylation of PS with oxidized methylene group inthe 15¹ position into purpurin 5 take place:

When chlorin e₆ turning into the “Liquid extract of chlorins” the use ofthe temperature less than 40° C. requires long reaction time, this beingtechnologically irrational. The use of the temperature higher than 100°C. results in the acceleration of substance decomposing.

Duration of the process less than for 1 hour requires the use oftemperatures above 100° C., or results in the substance having a lowbiological activity.

Duration of the process greater than for 30 days is accompanied byirreversible change (decomposing) of substance.

The optimal process temperature is 45-70° C. (FIG. 1).

The optimal process duration is 2-9 days at 70° C. (FIG. 2) or 1-48hours at 100° C. (FIG. 3), resulting in 5-20% of purpurine 5 in anadmixture.

The substance containing 5-20% of purpurine 5 and 80-95% of chlorin e₆in composition of active agent (PS) is suitable for producingwater-soluble injection medicinal forms. If the substance contains lessthan 5% of purpurine 5 it has a low biological activity. If thesubstance contains more than 20% of purpurine 5 its water solubilityworsens, that unfavorably affects the stability of medicinal forms atstorage and worsens the ability to filtration through microporousfilters. The last property is necessary for sterilisation of medicinalforms as tetrapyrrols medicinal forms cannot be sterilised by heating orUV irradiation because of high probability of undesirable chemicalchanges.

80-95% chlorin e₆ contents in the substance is necessary for keepingpurpuine 5 in water-soluble state.

The pH interval results from the fact that its lower value—pH 7.5—is thelower limit of chlorins solubility in water solutions resulting inconcentrations suitable for pharmaceutical utilisation, without addingsolubilizers. The upper limit—pH 8.5—is the limit of biologicaltolerance of hydroxide ions, [OH⁻].

The interval of chlorin e₆ concentrations 6,5-7,5% results from the useof technological methods of centrifugation or filtration at the stage ofseparating chlorin e₆ precipitate, these methods giving the productwithin this range of concentrations.

The invention is illustrated by drawings, on which FIG. 1 relates to themethod and shows formation of purpurine 5 depending on temperature atincubation for 30 days; FIG. 2 shows the dependence of purpurine 5content on incubation time at the temperature 70° C.; FIG. 3 shows thedependence of purpurine 5 content on incubation time at the temperature100° C.; FIG. 4 illustrates the pharmacokinetics of “Liquid extract ofchlorins” substance, used as the medicinal form “Radachlorin, 0.5%solution for injections” (“Photochlorin”) at tumorous mice atintravenous introduction in the dose of 20 mg/kg; FIG. 5 a shows thepresence of a chlorin e₆ metabolite (formula I), namely purpurine 5(formula II), in blood, and the curves marked as “1”, are taken for PSin 0,01 M borate buffer with pH 9.18, and the curves marked as “2”, aretaken for PS in blood; FIG. 5 b confirms the chlorin e₆ metabolism(formula I) into purpurine 5 (formula II) in liver; FIG. 6 shows the PMRspectrum of “Liquid extract of chlorins” substance, obtained in Example2; FIG. 7 shows the mass spectrum of “Liquid extract of chlorins”substance, obtained in Example 2; FIG. 8 shows the absorption visiblespectrum of “Liquid extract of chlorins” substance, obtained in Example2, the spectrum is taken in ethanol, substance concentration is 5mkg/ml; FIG. 9 shows the PMR spectrum of chlorin e₆; FIG. 10 shows themass spectrum of chlorin e₆; FIG. 11 shows the absorption visiblespectrum of chlorin e₆, the spectrum is taken in ethanol, chlorin e₆concentration is 15 mkg/ml (Sore band—5 mkg/ml); FIG. 12 shows the PMRspectrum of purpurine 5; FIG. 13 shows the mass spectrum of purpurine 5;FIG. 14 shows the absorption visible spectrum of purpurine 5, thespectrum is taken in ethanol, purpurine 5 concentration is 15 mkg/ml(Sore band—5 mkg/ml); FIG. 15 shows the PMR spectrum of purpurine 5dimethyl ester; FIG. 16 shows the mass spectrum of purpurine 5 dimethylester; FIG. 17 shows the absorption visible spectrum of purpurine 5dimethyl ester, the spectrum is taken in ethanol, purpurine 5 DME is 15mkg/ml (Sore band—5 mkg/ml).

PS is illustrated by Example 1, examples of method realisation are givenin Examples 2, 3, special cases of method realisation are illustrated inExamples 4-9.

In respect to chemistry PS comprises three cyclic tetrapyrrols of thechlorin nature (with hydrogenated D ring)—chlorin e₆ (Formula I),purpurine 5 (Formula II, Example 10) and purpurine 18, which graduallyturns into chlorin p₆ in alkaline medium (at storage) (Formula III).

In respect to physical chemistry PS possesses the ability to absorblight in visible spectrum, resulting in PS photoactivation and thesubsequent relaxation of excited state with transfer of energy tomolecular oxygen and organic substrates dissolved in tissues. Thistransfer leads to oxidising and free-radical processes in biologicaltissues and their damage and the subsequent destruction (necrosis). Themost preferable excitation band for PDT is the long-wavelength band(Tab. 1) since penetrating power of light in biological tissuesincreases along with increase in wavelength. Thus, PS is capable todamage biological objects on depth up to 10 mm after excitation by lightwith wavelength 654-670 nm.

In respect to pharmaceutics PS is “Liquid extract of chlorins” substance(extracts are considered as liquid if the effective agent concentrationis less than 20%). The given substance is considered as extract due tothe necessity of its extraction from a biomass with the use of organicsolvents. TABLE 1 Absorption maximum positions and molecular extinctionof absorption values for a long-wave band of “Liquid extract ofchlorins” in different media. λ_(max), nm λ_(max), nm ε, M⁻¹cm⁻¹ ε,M⁻¹cm⁻¹ (0.01 M bo- (0.01 M borate buffer λ_(max), nm rate buffer, with1% human serum ε, M⁻¹cm⁻¹ PS pH 9.18) albumin, pH 7.2) (ethanol) “Liquidextract of 654.5 (28270) 662 (34200) 662 (34230) chlorins”

Compound of formula II possesses the ability to accumulate selectivelyin malignant neoplasms and infected focuses but it is weakly soluble inwater, and compound of formula I, alongside with expressed photodynamicactivity, is a solubilising agent for compound of formula II.

With respect to pharmacology (FIG. 4, Example 11) the uniqueness ofpharmacokinetic parameters is achieved by the fact that in an organismPS of formula (I) slowly turns into PS of formula (II), this processkeeps the concentration of the last at a constant level from the momentof introduction into the organism till the moment of excretion out of atumour, during a time interval sufficient for effective PDT realisation.After the suggested PS composition is introduced into the organism oftumorous mice it comes into a blood flow, and due to blood circulationmainly of the compound of formula I high and stable PSconcentration—0.27-0.32 μM, sufficient for effective PDT in the intervalof 0,5-4 hours, is achieved in the first 3 hours post introduction inthe area of a tumour. Within this period high contrast is achieved dueto the presence of 5-20% of the compound of formula (II) in acomposition, this compound possesses the ability to accumulate in atumour with high contrast, the maximum of accumulation falling on themoment of 3 hours post PS introduction into an organism of animals (theindex of contrast is 14.5 for skin and 2.9 for muscles). Within thistime the compound of formula (I) turns into the compound of formula (II)in the organism, providing high stable PS concentration in the area of atumour in the interval of 3-5 hours after injection, this concentrationgradually decreases, remaining therapeutically sufficient up to 18 hoursafter injection. Then the compound of formula (II) dissociates in theorganism to nontoxical products that are excreted through a liver.

Chlorin e₆ of formula (I) transformation into purpurine 5 of formula(II) is proved by fluorescence spectra of experimental animals' organsand tissues samples (FIG. 5). When adding the “Radachlorin, 0.5%solution for injections” (“Photochlorin”) medicinal form to bloodhomogenate [FIG. 5 a, (1)] in the concentration of 10 μM with thesubsequent spectrophotometric analysis the change of fluorescencespectrum in the form of 1.2 times widening and fluorescence intensitymaximum shift to a long-wave spectral region by 8 nm is observed. Whenadding “Photochlorin” in a smaller concentration (C=1 μM) only the shiftof a spectrum without widening is observed, that shows the dose effectat metabolite formation [FIG. 5 a, (2)].

FIG. 5 a, (3) shows the result of blood homogenate analysis, received 3hours after “Photochlorin” introduction into mice. The most expressedparameter here is 1.5 times widening of a spectrum at the maximum shiftof a band to a long-wave spectral region by 4 nm, this indicating that amixture of “Photochlorin” and metabolite is present in the analysedblood sample.

When adding “Photochlorin” in concentration of 10 μM to a test tube withliver homogenate [FIG. 5 b, (1)] the change of spectral characteristicsis expressed first of all in shift of fluorescence intensity maximum toa long-wave spectral region by 9 nm. The widening of a spectrum is notobserved.

The similar picture is observed when adding “Photochlorin” in smallerconcentration C=1 μM [FIG. 5 b, (2)].

When liver tissue homogenate obtained 3 hours after the preparationintroduction into animals is analysed the spectrophotometric picture ofa sample is similar to two previous [FIG. 5 b, (3)].

Thus, the received data show the “Photochlorin” metabolite presence inliver homogenates.

The purpurine 5 accumulation in tumours of experimental animals, optimalfor photodynamic effect according to selectivity, is observed within thetime interval of 3-18 hours after intravenous or intraperitonealintroduction. In cases when it is necessary to enable also the chlorinssubstance circulating in a blood flow, the irradiation optimum time is0,5-4 hours after intravenous introduction. Generally, for realising PDTwith “Liquid extract of chlorins”, the interval between introduction ofa preparation and irradiation makes 0,5-18 hours.

Biological activity of the medicinal form “Radachlorin, 0.5% solutionfor injections” (“Photochlorin”), containing 0.5% of anhydrous “Liquidextract of chlorins” substance, is estimated in vitro and in vivo.

PS balance according to amphiphilicity is proved by standard experimentin vitro (Kessel D. Biochemistry. 1977. V. 16. p. 3443-3449) (Tab. 2,Example 12). PS distribution coefficient in 1-octanol/phosphate buffer,pH 7.4 (Cd) is 1.40. It means that the claimed PS is equally wellsoluble both in aqueous and in lipidic phase and it proves PS lipophilywhich allows this compound to be redistributed from water into complexeswith transport proteins and lipoproteins, to penetrate rapidly intocells and to accumulate in cytoplasmic intracellular membranes andmicrosomes, or to penetrate into cells by diffusion through a plasmaticmembrane of these cells. After laser irradiation the compound depositedin such a way evolves singlet oxygen inside a cell and kills it.

PS antitumor activity towards different types of cancer cells is provedby the results obtained in in vitro experiment in which 3 lines ofcultured tumor cells: rat pheochromocytomes PC12, rat Gasser's ganglionneurinoma RGGN1 and rat hepatoma 27 (Hep27), were used (Tab. 2, Example13).

The following methods are used for study of dose-dependentcytophototoxic (after laser irradiation) and biological “dark” activityof PS:

-   1. MTT-test that allows to define precisely the number of living    cells after their PS treatment and laser irradiation in order to    calculate cytotoxic and cytophototoxic indexes of PS. The same test    allows to estimate dose-dependent cytotoxic and biological “dark”    activity of PS (Andrei V. Reshetnickov, Gelii V. Ponomarev,    Andrei V. Ivanov, Olga Yu. Abakumova, Tatyana A. Tsvetkova,    Artashes V. Karmenyan, Aleksei G. Rebeko, Rudolf Ph. Baum. Novel    drug form of chlorin e₆//In Optical Methods for Tumor Treatment and    Detection: Mechanisms and Techniques in Photodynamic Therapy    IX.—T. J. Dougherty, ed., Vol. 3909, 124-129 (2000)).-   2. Determination of number of cells after cell monolayer staining    with crystal violet at the end of experiment. This method is less    laborious and expensive than MTT-test and it also allows to    calculate cytotoxic and cytophototoxic indexes of PS (A. E. Medvedev    et al., Biomed. Science, 1990, v.1, p. 261), but it is less precise    since crystal violet stains dead cells as well.-   3. Comparative genotoxic and genophototoxic effect of PS is    estimated by the degree of inhibition of DNA synthesis in cells. DNA    synthesis is evaluated by the level of incorporation of ¹⁴C    thymidine into DNA, using standard radiometric methods (O. Yu.    Abakumova, et. al., J. Neural. Transm. Suppl.3, 1998, V. 52, p. 87).

All of three studied cell lines are highly sensitive to laserirradiation effect after PS treatment (data of MTT-test). According tosusceptibility to laser irradiation cell lines range as follows:RGGN1>PC12>Hep27.

At prolonged PS effect in 5 μM concentration on cells in darknesssurvival was 96.5-86.2% for PC-12, 103.7-93.0% for RGGN1 and 109.7-87.9%for Hep27 (MTT-test-crystal violet, correspondingly). Under the sameconditions DNA synthesis in PC-12 cells stayed practically unaffectedand it was 21.2 and 22.2% reduced in Hep27 and RGGN1 cellscorrespondingly. The observable increase in number of RGGN1 and Hep27cells under effect of 5 μM PS on cells in darkness is most likelyrelated to induction of cells proliferative activity by PS. In generalcytotoxic activity is more typical for PS in the absence of irradiationthan induction of proliferative activity.

Cell death is observed after laser irradiation of cells treated with PS.Dose-dependent cytophototoxic activity of the preparation is detectedand it allows to calculate EC₅₀, i.e. to determine the PS concentrationat which 50% of cells die. These data are given in Table 2. It should benoted that PS with EC₅₀ less than 20 μM are considered to be efficientfor tumor growth suppression.

DNA synthesis in PC-12 cells is strongly decreased (96.5% decreasecomparing to only irradiated control) at determination ofgenophototoxicity after treating the cells with 5 μM PS and laserirradiation. DNA synthesis stimulation after laser irradiation at low PSconcentrations is observed in Hep27 and RGGN1 cells, this synthesisbeing considerably reduced in the presence of 5 μM RC. The observableDNA synthesis stimulation may be explained by the fact that transformedliver and glia cells that survived at low PS concentrations possess highability to synthesize DNA and to regenerate the population.

Thus, PS is a highly cytophototoxic preparation for different types oftumor cells. In high concentrations (>5 μM) it is a moderate inhibitorof tumor growth even without irradiation. Due to high genophototoxicityPS can be considered as a strong tumor growth inhibitor at irradiation.

PS toxic properties were studied in in vivo experiments (Example 14).The average LD₅₀ is 210.53±22.2 mg/kg weighting coefficient beingconsidered, and the dose causing the death of 10% of experimentalanimals (LD₁₀) is 169,87 mg/kg. These experiments allow to consider PSas a “Low toxic substance”.

PS biodistribution was studied in in vivo experiments (Example 11). Thefollowing mechanisms of distribution of compounds are observed when PSis introduced intraperitoneally to mice with T36 embryocarcinomainoculated into hind leg muscle. After injection PS gets into blood, andthen it is redistributed into organs and tissues of an animal (Tab. 3).

As it can be seen in Table 3 tumour accumulation maximum (0,70 μM) isachieved in 5 hours after intraperitoneal injection in a dose of 40mg/kg and it is conserved for a long period (18-24 hours). Tumoralconcentration 18 h after injection is 0.48 μM, that is 1,5 times lessthan in accumulation absolute maximum at high selectivity ofaccumulation. The tumour/muscle tissue ratio is 32, and tumour/skinratio is 44.

Tumour accumulation maximum (0,32 μM) is achieved in 0.5 hours afterintravenous injection in a dose of 20 mg/kg and it is also conserved fora long period (up to 5 hours). Maximum contrast of accumulation atintravenous injection is achieved in 3 hours and this value makes 3 fortumor/muscle tissue and 4 for tumor/skin. PS is excreted out of theorganism by 98% in a day. TABLE 2 Lipophily coefficient and in vitroactivity of “Radachlorin, 0.5% solution for injections”(“Photochlorin”). Cytotoxicity Photocyto- (“dark” toxicity), toxicity,Tests, cell lines % to control at 5 μM EC₅₀, μM¹ (Cd) MTT test, PC-1296.5 1.8 1.40 MTT test, RGGN1 103.7 1.8 MTT test, Hep27 109.7 3.9Crystal violet test, PC-12 86.2 1.5 Crystal violet test, RGGN1 93.0 1.8Crystal violet test, Hep27 87.9 4.7 Genotoxicity, PC-12 104.7 3.5 % tocontrol at 5 μM Genotoxicity, RGGN1 77.8 132.2 % to control at 5 μMGenotoxicity, Hep27 78.8 100.7 % to control at 5 μM¹Except for genophototoxicity

The results of the preparation effectiveness estimation at PDT of cancerin in vivo experiments on mice (Example 15) allow to state that“Radachlorin, 0.5% solution for injections” (“Photochlorin”) and“Radachlorin, 0.05% gel” possess the expressed photodynamic activity.

The “Liquid extract of chlorins” medicinal substance including chlorinssodium salts (or salts of chlorins and other strong inorganic bases) isused for producing medicinal forms by supplementing different additivesapproved by RF State Pharmacopoeia: calcium carbonate, saccharose,glucose, starch, magnesium stearate, polyvinylpirrolidones, polyglucans,methylglucamine, isotonic solution, dimethylsulfoxide, gel andwater-emulsion substrates etc. (Examples 4-9).

Ointments, liniments, gels, oil-based preparations are used for externaluse, these forms contain the substrates approved by RF StatePharmacopoeia, 5-20% of dimethylsulfoxide and 0.5-12% of “Liquid extractof chlorins” substance, or 0.8-14% of “Liquid extract of chlorins”substance and 86-99.2% of dimethylsulfoxide (Examples 8, 9).

Dimethylsulfoxide concentrations range in combination with substrates isexplained by the fact that substance penetration into tissues is low atconcentration less than 5%, and that reduces the PDT effectiveness. Ifdimethylsulfoxide concentration is higher than 20% medicinal forms onother bases lose their stability at storage. Substance concentrationsrange is explained by the fact that if this concentration is less than0.5% substance concentration in tissue is insufficient for effectivePDT. If substance concentration is higher than 12% tissue losestransparence for light radiation, all light is absorbed in the upperlayer of the tissue that results in a burn at low effectiveness of PDTprocedure. TABLE 3 The main pharmacokinetic parameters AbsoluteTumour/skin Tumour/mus- Tumour accumulation Tumour ratio at cles ratioat accumulation Tumour/skin Tumour/mus- maximum, accumulation tumourtumour at contrast ratio at cles ratio at organ-μM- maximum,accumulation accumulation maximum, contrast contrast Excretion, PS hourμM-hour maximum maximum μM-hour maximum maximum %---hour Photochlorinesmall 0.32-0.5 6.4 1.6 0.29-3 14.5 2.9 98---24 intravenous, intestines -20 mg/kg 4.0-0.5 Photochlorine blood - 0.70-5 3.9 3.0 0.48-18 44.0 32.098---24 intraperitoneally, 5.2-0.5 40 mg/kg

Substance exposition on skin before irradiation is 0.5-24 hours atexternal use. The substance has no time to penetrate into a tissue onnecessary depth in time less than 0,5 hours. If time interval is morethan 24 hours the fall of preparation absolute accumulation value isobserved due to its redistribution and excretion. Besides, long-timeexpositions of external medicinal forms on a skin are inconvenient fromthe clinical point of view.

The preparation is used for intravenous dropwise or stream introductionin the form of 0.1-1% solutions in any mediums approved by RF StatePharmacopoeia (apyrogenic water for injections, dimethylsulfoxide,saline solution, etc.). Use of solutions of the substance withconcentration less than 0.1% is irrational considering volumes of liquidintroduced into an organism. Use of solutions with concentration higherthan 1% is impossible due to the low filterability of such solutions ata stage of sterilisation through antibacterial filters.

Semiconductor laser diode module for photodynamic therapy ML-662-SPdesigned by ZAO “MYLON” (Saint Petersburg) and OOO “SIGM PLUS” (Moscow)is used for activation of PS “Liquid extract of chlorins” substance.This module has the following output data (Certificate of the RussianMinistry of Health, Reg. No. 29/10-679-96):

-   -   power of 2,5-3 W in a fibre of 200 microns with the aperture        0.22.    -   high intensive laser diodes of “Polaroid” corporation (USA) and        OOO “SIGM PLUS” coproduction with maximum irradiation wavelength        of 662±3 nm.

Modules of a lower power (with a lower number of diodes) with maximumirradiation wavelength of 662±3 nm may be used for activation of PSsubstance, the solid-state laser with pumping on a second harmonic ofyttrium-aluminium garnet YAG:Nd³⁺ with maximum irradiation wavelength of670 nm may be used also.

Magnitude of fed energy varies from 30 up to 3000 J. At light doses lessthan 30 J the PDT procedure becomes excessively long since scanningshould be realised at the extremely small areas in order to achieve theoptimal effect. At light doses more than 3000 J and tumour dimensionsmost frequently occurring in clinical practice the considerable damageof healthy tissue leading to prolongation of regeneration period isobserved.

Surface density of fed energy varies from 50 up to 2500 J/cm². Atsurface light doses less than 50 J/cm² no effect is observed. At surfacelight doses more than 2500 J/cm² the considerable damage of healthytissue leading to prolongation of regeneration period is observed.

The range of wavelengths of exciting radiation is connected to atechnical characteristic of the used laser (662±3 nm), shift of thepreparation absorption maximum depending on polarity of medium (654-662nm) and the content of purpurine 5 in the substance (5-20%, half-widthof a long-wavelength absorption band at 663-670 nm) (Tab. 1).

EXAMPLE 1 Description of the Physicochemical Properties of PS

PS represents dense black mass, acquiring a green shade in a thin layer,with an odour of algae.

In order to confirm authenticity of PS properties “Liquid extract ofchlorins”, 7.5% is thoroughly stirred, a portion of the extract (1 mg)is dissolved in 10 ml of medical or the most purified rectified ethylalcohol, 95%, and optical density is measured at 662 nm (D). The valueis 0,23. The molecular extinction ε (M⁻¹cm⁻¹) is calculated according tothe formula ε=D*597/(0,004). The resulting value should lie within therange of 33300-35100. After substitution, ε=0,23*597/(0,004)=34328.Hence, “Liquid extract of chlorins” contains 7.5% PS.

PS solution in ethyl alcohol has yellow-green colour. The solutionacquires ruby-red colouring if light rays from the medical blue lamp MDS220-75 (technical specifications 16.535.376-79) are passed through thesolution layer in the light-protected place.

For the quantitative determining “Liquid extract of chlorins” isthoroughly stirred, a portion of the extract (5 mg) is dissolved in 10ml of medical or the most purified rectified ethyl alcohol, 95%, andoptical density is measured at 662 nm (D). The value is 2,15. PS contentis calculated according to the formula: c,%=(D*597*10*100)/(34230*5).The resulting value should correspond to the specified. Aftersubstitution, c,%=(2,15*597*10*100)/(34230*5)=7,5% (corresponds to thespecified).

For the further analyses dilute hydrochloric acid solution is added to100 mg of “Liquid extract of chlorins” till PS precipitates, theprecipitate is filtered out, dried in vacuum over phosphorus pentoxidefor 12 h and PMR-, mass spectrums and absorption spectrum are taken inthe wavelength range of 360-720 nm.

PS PMR spectrum (FIG. 6): (in DMSO-D6, conc. solution): 9.64, 9.55,9.52, 9.39, 8.90, 8.79 (s, meso-H of chlorin e₆ and purpurine 5), 8.09,8.04, 7,97, 7.92 (2d, CH═CH₂ of chlorin e₆ and purpurine 5), 6.84 (s,γ-meso-CHO of purpurine 5), 6.37, 6.32, 6.13, 6.10 (2d, CH═CH ₂), 5.43(2s, γ-meso-CH ₂COOH), 4.60 (m, 7-H), 4.45 (m, 8-H), 3.80, 3.56(q×2,4-CH ₂CH₃), 3.75, 3.64, 3.51, 3.46, 3.29, 3.23 (c, nuclear CH ₃ ofchlorin e₆ and purpurine 5), 2.38, 2.32 (2m, 7-CHH₂CH₂COOH), 2.71, 2.20(2m, 7-CH₂CH ₂COOH), 1.76 (d, 8-CH ₃), 1.72 (t, 4-CH₂CH ₃),-1.63,-1.91(2s, 2NH) ppm.

PS mass spectrum (FIG. 7): e.i., M⁺ (%), 596 (16.0), 566 (9.4), 508(100.0), 494 (7.3), 447 (9.4), 435 (50.6), 421 (12.8), 405 (6.9), 254(7.4).

PS visible absorption spectrum: λ (ε) (ethanol), 386 (22310), 406(113040), 506 (14870), 536 (8925), 608 (7437), 662 (34220).

According to PMR spectrum the substance contains 80% of chlorin e₆, 15%of purpurine 5 and 5% of purpuine 18 (minor signals at 9.25, 9.10, 8.71,7.84, 3.55, 3.32, 3.04 ppm), that corresponds to a composition beingpatented. According to the mass spectrum there are peaks of molecularions 596 of chlorin e₆ and 566 of purpurine 5. In absorption spectrumthere is a band of 662 nm with the absorption value that is wellmatching the molecular extinction of PS etalon (34230).

Hence, the studied sample is “Liquid extract of chlorins”, 7.5%.

EXAMPLE 2 Producing PS as “Liquid Extract of Chlorins”, 6.5%

Spirulina biomass (2 kg) is treated with acetone (3×2 L) tillchlorophyll a is completely extracted, the biomass is filtered out, theextract is treated with hydrochloric acid (30 ml) in order to removemagnesium ion out of the chlorofyll molecule, the extract is neutralisedand precipitated pheophytin a (8 g) is filtered out, then pheophytin ais hydrolysed in the mixture of hydrochloric acid-acetone-hexane, forthis purpose pheophytin a is dissolved in the mixture of 50 ml ofacetone, 5 ml of hexane and 40 ml of hydrochloric acid (37%), themixture is heated up to 40° C. and stirred for 1 hour, then hexane (50ml) is added and organic phase is washed with the mixture of acetone andhydrochloric acid (2:1, 3×50 ml), water phase is washed with hexane(5×40 ml), then water phase containing pheophorbid a is neutralised withexcess of sodium citrate (tri-, di- or mono-substituted) water solution,precipitated pheophorbid a is filtered out, washed with water (3×50 ml),recrystallised out of the acetone-water mixture, air dried till its massbecomes constant (pheophorbid a yield is 4.2 g, 7.1 mM, 77%), thenpheophorbid a (2.7 g, 4.56 mM) is dissolved in acetone (100 ml), stronginorganic base is added in the form of water solution (0.05%, 25 ml),stirred at 60° C. for 5 min, extra volume of inorganic base is added inthe form of water solution (20%, 25 ml), the mixture is heated at 40° C.for 90 min, neutralised with diluted hydrochloric acid (2%, about 250ml), chlorin e₆ precipitate is separated by centrifugation, washed withdistilled water (5×10 ml) till acid reaction disappears, 1.85 g (2.96mM, 65%) chlorin e₆ is obtained, then chlorin e₆ is recrystallised outof acetone in order to separate linear tetrapyrrols, chlorin e₆ isfiltered out and washed three times with distilled water, chlorin e₆ isheated in sealed reservoir at the temperature of 40° C. for 30 days,then it is cooled and 1% sodium hydroxide solution is added till pH 7.5,the resulting PS contains 15% of purpurine 5, 80% of chlorin e₆ and 5%of purpurine 18 (chlorin p₆), then PS solution is adjusted withdistilled water to make photosensitizer concentration 6.5%, giving 14.2g (50%) PS in the form of 6.5% “Liquid extract of chlorins”.

The resulting “Liquid extract of chlorins” PMR spectrum (FIG. 6): (inDMSO-D6, conc. solution): 9.64, 9.55, 9.52, 9.39, 8.90, 8.79 (s, meso-Hof chlorin e₆ and purpurine 5), 8.09, 8.04, 7.97, 7.92 (2d, CH═CH₂ ofchlorin e₆ and purpurine 5), 6.84 (s, γ-meso-CHO of purpurine 5), 6.37,6.32, 6.13, 6.10 (2d, CH═CH ₂), 5.43 (2s, γ-meso-CH ₂COOH), 4.60 (m,7-H), 4.45 (m, 8-HH), 3.80, 3.56 (q×2,4-CH ₂CH₃), 3.75, 3.64, 3.51,3.46, 3.29, 3.23 (s, nuclear CH ₃ of chlorin e₆ and purpurine 5), 2.38,2.32 (2m, 7-CH ₂CH₂COOH), 2.71, 2.20 (2m, 7-CH₂CH ₂COOH), 1.76 (d, 8-CH₃), 1.72 (t, 4-CH₂CH ₃),-1.63,-1.91 (2s, 2NH) ppm.

The substance contains 80% of chlorin e₆, 15% of purpurine 5 and 5% ofpurpurine 18 (chlorin p₆) (signals at 9.25, 9.10, 8.71, 7.84, 3.55,3.32, 3.04 ppm).

The resulting substance mass spectrum (FIG. 7): e.i., M⁺ (%), 596(16.0), 566 (9.4), 508 (100.0), 494 (7.3), 447 (9.4), 435 (50.6), 421(12.8), 405 (6.9), 254 (7.4).

Visible absorption spectrum (FIG. 8): λ(ε) (ethanol), 386 (22320), 406(113110), 506 (14880), 536 (8930), 608 (7440), 662 (34230).

EXAMPLE 3 Producing PS as “Liquid Extract of Chlorins”, 7.5%

Spirulina biomass (2 kg) is treated with acetone (3×2 l) tillchlorophyll a is completely extracted, the biomass is centrifuged out,the extract is treated with hydrochloric acid (30 ml) in order to removemagnesium ion out of the chlorofyll molecule, the extract is neutralisedand precipitated pheophytin a (8 g) is filtered out, then pheophytin ais hydrolysed in the mixture of hydrochloric acid-acetone-hexane, forthis purpose pheophytin a is dissolved in the mixture of 100 ml ofacetone, 50 ml of hexane and 80 ml of hydrochloric acid (37%), themixture is heated up to 60° C. and stirred for 20 min, then hexane (100ml) is added and organic phase is washed with the mixture of acetone andconcentrated hydrochloric acid (5:1, 3×50 ml), water phase is washedwith hexane (5×40 ml), then water phase containing pheophorbid a isneutralised with excess of sodium citrate (tri-, di- ormono-substituted) water solution, precipitated pheophorbid a is filteredout, washed with water (3×50 ml), recrystallised out of theacetone-water mixture, air dried till its weight becomes constant (yieldis 3.8 g, 6.4 mM, 67%), then pheophorbid a (2.7 g, 4.56 mM) is dissolvedin acetone (100 ml), strong inorganic base is added in the form of watersolution (1%, 25 ml), stirred at 30° C. for 30 min, extra volume ofstrong inorganic base is added in the form of water solution (20%, 25ml), the mixture is heated at 60° C. for 20 min, neutralised withdiluted hydrochloric acid (2%, about 250 ml), chlorin e₆ precipitate isseparated by centrifugation, washed with distilled water (5×10 ml) tillacid reaction disappears, 1.67 g (2.67 mM, 55%) chlorin e₆ is obtained,then chlorin e₆ is recrystallised out of acetone in order to separatelinear tetrapyrrols, chlorin e₆ is filtered out and washed three timeswith distilled water, chlorin e₆ is heated in sealed reservoir at thetemperature of 100° C. for 1 hour, then it is cooled and 1% potassiumhydroxide solution is added till pH 8.5, the resulting PS contains 2% ofpurpurine 5, 82% of chlorin e₆ and 16% of purpurine 18 (chlorin p₆),then PS solution is adjusted with distilled water to makephotosensitizer concentration 7.5%, giving 11.1 g (50%) PS in the formof 7.5% paste.

The resulting substance spectra are similar to those given in Example 2and represent a superposition of spectra of chlorin e₆ (FIG. 9-11) andpurpurine 5 (FIG. 12-14).

EXAMPLE 4 A Special Case of Producing PS—Producing “Liquid Extract ofChlorins”, 7.5%

PS containing 2% of purpurine 5, 82% of chlorin e₆ and 16% of purpurine18 (chlorin p₆) in the form of 7.5% paste described in the previousExample is gel filtrated on a Sephadex G10 column of 50 mm diameter and100 mm height with the use of 1% potassium hydroxide solution as aneluent, till chlorin e₆ content becomes 90%, purpurine 5-5% andpurpurine 18-5%. Diluted hydrochloric acid solution is added till PSprecipitates, PS is adjusted with apyrogenic water for injections tomake photosensitizer concentration 7.5% _(mass) giving 6.8 g “Liquidextract of chlorins”, 7.5%. An electron spectrum of the product—see.FIG. 8.

EXAMPLE 5 A Special Case of Producing PS-Producing the “Radachlorin,0.1% Solution for Injections” Medicinal Form

After gel filtration diluted hydrochloric acid solution is added to thesolution of PS described in Example 4 till PS precipitates, thisprecipitate is filtered out, concentrated sodium hydroxide solution inapyrogenic water for injections is added till pH 7,5 and apyrogenicwater for injections is added to make PS concentration 0.1%, thenbacteria are filtered off the solution through antibacterial “Millipore”microporous filter with 0,22 μm pores. The yield is 500 ml of thesolution. An electron spectrum of the product—see. FIG. 8.

EXAMPLE 6 A Special Case of Producing PS-Producing the “Radachlorin,0.5% Solution for Injections” (“Photochlorin”) Medicinal Form

After gel filtration diluted hydrochloric acid solution is added to thesolution of PS described in Example 4 till PS precipitates, thisprecipitate is filtered out, concentrated potassium hydroxide solutionis added till pH 7, then the solution is adjusted withN-methyl-D-glucamine up to pH 8,5 under pH-meter control, apyrogenicwater for injections is added to make photosensitizer concentration 0.5%_(mass), then bacteria are filtered off the solution throughantibacterial “Millipore” microporous filter with 0,22 μm pores. Theyield is 100 ml of the solution. An electron spectrum of theproduct—see. FIG. 8.

EXAMPLE 7 A Special Case of Producing PS-Producing the “Radachlorin, 1%Solution for Injections” Medicinal Form

After gel filtration diluted hydrochloric acid solution is added to thesolution of PS described in Example 4 till PS precipitates, thisprecipitate is filtered out, concentrated sodium hydroxide solution isadded till pH 8,5 then apyrogenic water for injections is added to makephotosensitizer concentration 1% _(mass), then bacteria are filtered offthe solution through antibacterial “Millipore” microporous filter with0,22 μm pores. The yield is 50 ml of the solution. An electron spectrumof the product—see. FIG. 8.

EXAMPLE 8 A Special Case of Producing PS-Producing the “Radachlorin,Gel” Medicinal Forms

After gel filtration diluted hydrochloric acid solution is added to thesolution of PS described in Example 4 till PS precipitates, thisprecipitate is centrifuged out, adjusted with apyrogenic water forinjections to make photosensitizer concentration 6.5% _(mass), then thefollowing variants are realised:

Variant (a). 0,3 g of Pemulen TR1 or Carbopol 2020 (BF Goodrich, UK) areadded to 75 ml of water and 5 g of dimethylsulfoxide at room temperatureand stirred for ¼-8 hours. Water alkaline solution is added till pH 5.Gel is resuspended supplementing “Liquid extract of chlorins”, 6.5% andwater to make 0.05% concentration of chlorin e₆ in resulting gel, gel isvacuumised for 5 minutes at 10-50 mm Hg. The yield is 100 g of the gel.

Variant (b). 5 g of dimethylsolfoxide and “Liquid extract of chlorins”,6.5% is added to 70 ml of water to make 0.05% concentration of chlorine₆ in resulting gel, then 15 g of Aculyn 33A (ISP, USA) is added. Thesubstance is stirred to homogeneity and water alkaline solution is addedtill pH 5. Gel is vacuumised for 5 minutes at 10-50 mm Hg. The yield is100 g of the gel.

After gel filtration diluted hydrochloric acid solution is added to thesolution of PS described in Example 4 till PS precipitates, thisprecipitate is centrifuged out, adjusted with apyrogenic water forinjections to make photosensitizer concentration 7.5% _(mass), then thefollowing variants are realised:

Variant (c). 0,7 g of Pemulen TR1 or Carbopol 2020 (BF Goodrich, UK) areadded to 60 ml of water and 20 g of dimethylsulfoxide at roomtemperature and stirred for ¼-8 hours. Triethanolamine water solution isadded till pH 8.5. Gel is resuspended supplementing “Liquid extract ofchlorins”, 7.5% and water to make 1% concentration of chlorin e₆ inresulting gel, gel is vacuumised for 5 minutes at 10-50 mm Hg. The yieldis 100 g of the gel.

Variant (d). 20 g of dimethylsolfoxide and “Liquid extract of chlorins”,7.5% is added to 55 ml of water to make 1% concentration of chlorin e₆in resulting gel, then 15 g of Aculyn 33A (ISP, USA) is added. Thesubstance is stirred to homogeneity and triethanolamine water solutionis added till pH 8.5. Gel is vacuumised for 5 minutes at 10-50 mm Hg.The yield is 100 g of the gel.

EXAMPLE 9 A Special Case of Producing PS-Producing the “Radachlorin,Dimethylsulfoxide Solution for External Use” Medicinal Forms

Variant (a). After gel filtration in Example 4 diluted hydrochloric acidsolution is added to the mixture till PS precipitates, this precipitateis filtered out, adjusted with apyrogenic water for injections to makePS concentration 7.5% _(mass), and 14 g of the resulting “Liquid extractof chlorins” is added to 86 g of dimethylsulfoxide at room temperatureto make 1% concentration of chlorin e₆ in the resulting solution, andstirred to homogeneity. The yield is 100 g of the solution.

Variant (b). After gel filtration in Example 4 diluted hydrochloric acidsolution is added to the mixture till PS precipitates, this precipitateis filtered out, adjusted with apyrogenic water for injections to makePS concentration 7.5% _(mass), and 0.8 g of the resulting “Liquidextract of chlorins” is added to 99.2 g of dimethylsulfoxide at roomtemperature to make 0.05% concentration of chlorin e₆ in the resultingsolution, and stirred to homogeneity. The yield is 100 g of thesolution.

EXAMPLE 10

For purpurine 5 identification the reaction mixture of Example 2 is gelfiltrated on a Sephadex G10 column with the use of 1%N-methyl-D-glucamine solution as an eluent to give 3 fractions, thefirst and the second fractions contain purpurine 5. These fractions areneutralised, a precipitate is filtered out, dissolved inchloroform-methanol 1:1 mixture and esterified with diazomethane. Themixture is washed with water, an organic phase is separated, dried withanhydrous magnesium sulfate, concentrated by evaporation in vacuum andchromatographed on silica gel Merck, Kieselgel, 0,04-0,063, the last(least mobile) fraction is collected. If necessary the resultingpurpurine 5 dimethyl ester (10,1% calculating to the dry reaction masstaken for esterification) is chromatographed repeatedly.

PMR spectrum (FIG. 15): (DMSO-D6, conc. solution): 9.64, 9.46, 8.82 (s,meso-H), 8.06 (2d, CHH═CH₂), 6.82 (s, γ-meso-CHO), 6.34, 6.31, 6.19,6.16 (2d, —CH═CH ₂), 4.54 (m, 7-H), 4.46 (m, 8-H), 3.61 (q, 4-CH ₂CH₃),4.20, 3.81, 3.57, 3.53, 3.47 (5s, —COOCH ₃ and nuclear —CH ₃), 2.38,2.35 (2m, 7-CH ₂CH₂COOH), 2.68, 1.85, (2m, 7-CH₂CH ₂COOH), 1.73 (d, 8-CH₃), 1.70 (t, 4-CH₂CH ₃) ppm.

Mass spectrum (FIG. 16): e.i., M⁺ (%), 594 (8.6), 566 (100.0), 505(5.1), 491 (9.8), 475 (8.2), 463 (1.7), 447 (1.4), 433 (1.7), 403 (2.0),262 (5.0).

Visible absorption spectrum (FIG. 17): λ(ε) (chloroform), 408 (117200),501 (11380), 542 (9830), 617 (6720), 668 (35200).

Purpurine 5 dimethyl ester is dissolved in acetone and concentratedhydrochloric acid (37%) is added in the ratio 1:2. The mixture isstirred for 2 hours at 25° C., neutralised, purpurine 5 is filtered out,washed with water, dissolved in 10% N-methyl-D-glucamine solution andgel filtrated on a Sephadex G10 column with the use of 1%N-methyl-D-glucamine solution as an eluent, the second fraction iscollected, neutralised, a precipitate is filtered out, washed withwater, dried over phosphorus pentoxide till the weight becomes constantto give purpurine 5 (5,2% calculating to the dry reaction mass taken foresterification).

PMR spectrum (FIG. 12): (DMSO-D6, conc. solution): 9.55, 9.39, 8.79 (s,meso-H, 8.09, 8.04, 7.97, 7.92 (2d, —CH═CH₂), 6.84 (s, γ-meso-CHO),6.37, 6.32, 6.13, 6.10 (2d, —CH═CH ₂), 4.60 (m, 7-H), 4.45 (m, 8-H),3.55 (q, 4-CH ₂CH₃), 3.75, 3.46, 3.23 (s, nuclear-CH ₃), 2.38, 2.32 (2m,7-CHH₂CH₂COOH), 2.71, 2.20 (2m, 7-CH₂CH ₂COOH), 1.76 (d, 8-CH ₃). 1.72(t, 4-CH₂CH ₃) ppm.

Mass spectrum (FIG. 13): e.i., M⁺ (%), 566 (8.2), 494 (100.0), 447(9.1), 435 (49.6), 421 (12.7), 405 (6.6), 254 (7.1).

Visible absorption spectrum (FIG. 14): λ (ε) (ethanol), 408 (116900),501 (11320), 540 (9790), 615 (6710), 665 (35090).

EXAMPLE 11 Study of Pharmacokinetics and Metabolism of the “LiquidExtract of Chlorins” Substances and the “Radachlorin, 0.5% Solution forInjections” (“Photochlorin”) Medicinal Form

769,2 mg/kg of 6.5% “Liquid extract of chlorins” substance from theExample 2 (50 mg/kg calculating to anhydrous chlorins substance) wereintroduced intraperitoneally to mice of the line Balb/c. The mice wereslaughtered 3 hours after injection (each group consisted of 3 mice).The materials of liver, kidney, spleen, lungs, small intestines, tumor,surrounding muscle tissue as well as from blood, urine, faeces fromlarge intestine weighing 100 mg each were thoroughly homogenised inglass homogenizers supplementing 4 ml of saline solution. Forexamination of biological fluids (blood, urine) 0,1 ml of each fluidwere taken with subsequent dissolution in 4 ml of saline solution. Theresulting homogenates were studied on “Perkin-Elmer” spectrofluorimeter(MPF-44A model).

Study of the “Radachlorin, 0.5% solution for injections”(“Photochlorin”) medicinal form was carried out in a similar way inhomogenates of organs and tissues of mice to whom the preparation wasintroduced intraperitoneally in the dose of 50 mg/kg, the animals wereslaughtered 3 h after injection.

In both cases there is a shift of fluorescence intensity maximum intissues of liver, small intestines, spleen and kidney to 670 nm (by10-12 nm comparing to 0,01M borate buffer solution, pH 9.2, and by 5-6nm comparing to 0,01M borate buffer solution, pH 9.2, with 1% of humanserum albumine), that indicates the metabolism of “Radachlorin, 0.5%solution for injections” (“Photochlorin”) (FIG. 5).

In fluorescence spectrum this phenomenon looks differently, than asimple spectrum widening and shift to the long-wavelength range due toeffect of hydrophoby of medium (for example, after a hydrophobicinteraction with proteins, lipoproteins). Shift of intensity maximum isobserved without or with a little widening of a band that is typical offormation of a new compound. Fluorescence spectra of purpurine 5 in0,01M borate buffer solution, pH 9.2, with 1% of human serum albumin arecharacterised by presence of 670 nm band.

In blood, pulmonary parenchyma, as well as in skin and tumour thewidening of spectra 1,4-1,5 times at wavelength of 669 nm is observed,that indicates the presence of “Radachlorin, 0.5% solution forinjections” (“Photochlorin”) (its complex with proteins) and ametabolite mixture in homogenates.

If “Radachlorin, 0.5% solution for injections” (“Photochlorin”) is addeddirectly to the tubes with homogenates of intact animals tissues inconcentrations of 0.5-1.0 μM a metabolite of “Radachlorin, 0.5% solutionfor injections” (“Photochlorin”) is detected in blood, small intestines,liver, spleen and lung (a shift to long-wavelength range withoutwidening of a spectrum), and only in skin homogenate a slight 1,15 timesincrease in spectrum half-width is observed, that indicates the presenceof “Photochlorin”—metabolite mixture in a sample.

If “Radachlorin, 0.5% solution for injections” (“Photochlorin”)concentration in homogenates of organs is increased to 5-10 μM, thepresence of chlorin e₆-purpurine 5 mixture is registered practically inall the saples (a shift of spectra to long-wavelength range at 1,15-1,05times increase in half-width of spectra).

Thus, it is possible to consider that formation of a metabolite at“Radachlorin, 0.5% solution for injections” (“Photochlorin”) addition tohomogenates depends on concentration of the preparation and activity ofenzymes of a homogenised tissue.

These experiments clearly demonstrate the conversion of chlorin e₆ intopurpurine 5 under in vivo and ex vivo conditions. This conversion issimilar to the conversion of chlorin e₆ into purpurine 5 at heating.

EXAMPLE 12 N-Octanol/Phosphate Buffer, pH 7.4, Distribution Coefficient

300 ml of n-octanol and 300 ml of phosphate buffer, pH 7.4, are vortexedfor 20 sec and centrifuged for 10 min at 10000 rpm for splitting. 0.1 mlPS aliquot with PS concentration of 5 mg/ml is dissolved in preparedbuffer solution (2 ml) and n-octanol (8 ml), absorption maximum isdetermined at 406 nm.

The values of D^(o) _(c), and D^(b) _(c) are obtained, where o isn-octanol, b is phosphate buffer, c is control. Equilibriumn-octanol/phosphate buffer distribution is achieved by vortexing of 2 mlof phosphate buffer and 8 ml of n-octanol with 0.1 ml of PS for 20 secat 20° C. with subsequent centrifugation for 10 min at 10000 rpm.Optical density of each phase is measured at 406 nm giving the values ofD^(o) and D^(b), where o is n-octanol, b is phosphate buffer.

C_(d) is calculated according to the formula:

-   -   C_(d)=(D^(o) V^(o) D^(o) _(c) V^(o) _(c))/(D^(b) V^(b) D^(b)        _(c) V^(b) _(c)), where V^(o) _(c) —the volume of octanol taken        for determination of equilibrium distribution (8 ml), V^(o)        _(c)—the volume of octanol, saturated with water, taken for        control determination of aliquot absorption (8 ml), V^(b)        _(c)—the volume of the buffer taken for determination of        equilibrium distribution (2 ml), V^(b) _(c)—the volume of the        buffer, saturated with octanol, taken for control determination        of aliquot absorption (2 ml). Experiment is carried out for        three times and the obtained values of C_(d) are averaged.

The resulting value is 1.4±0.3.

EXAMPLE 13 Determination of In Vitro Phototoxicity (Biological Activity)and Cytotoxicity (Cell Toxicity) of the “Radachlorin, 0.5% Solution forInjections” (“Photochlorin”) Medicinal Form

For this work the laminar “Flow Lab” (UK), the CO₂-incubator “Flow Lab”(UK), the multiscan “Bio-Tek Instruments” (USA), mediums and serums“PanEco” (Russia) are used.

For one experiment cells of one line are passed in two 48-cell plates:one for laser irradiation and one for the “dark” experiment. Next daythe preparation is added to the confluent state cells and the plates arethermostated in a black paper. The preparation concentrations of 0.1,0.5, 2.0 and 5.0 μM are studied. 3 hours after addition of thepreparation the cells are irradiated with laser, the expositionirradiation dose being 50 J/cm², and 39 hours later the MTT-test iscarried out as well as incubation with ¹⁴C-thymidine for estimation ofDNA synthesis (the “dark” tray is tested too). In all the cases theupper part of the plate is used for the MTT-test, and the lower part isused for measurement of DNA synthesis and number of cells after stainingwith crystal violet.

The data represented in Tab. 2 are the mean of 4 parallel experiments.

EXAMPLE 14 In Vivo Study of Toxic Properties of the “Liquid Extract ofChlorins” Substance and the “Radachlorin, 0.5% Solution for Injections”(“Photochlorin”) Medicinal Form

Toxicity is studied at intravenous injection of PS to laboratory whitemice weighing 19-21 g (nursery of the Russian Academy of MedicalSciences, Krukovo). The animals are kept under standard vivariumconditions and are fed according to the Ministry of Health of the USSROrder No 1179 of 10.10.83 “About the approval of specifications offorage expenditures for laboratory animals in health protectioninstitutions”. Toxicity is determined according to the animal death,after calculation of the mean lethal dose—LD₅₀. The calculation iscarried out according to statistical methods recommended by the StatePharmacopoeia, edition XI (1,3). On the basis of LD₅₀ the studiedpreparation is referred to the specific class of toxicity according toHodge and Sterner. The intoxication reactions are also registered duringthe experiment.

12 mice (6 male and 6 female) are used for each PS dose being tested.The following doses are used for determination of PS LD₅₀: 5, 10, 15,20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275 mg/kg. Thesolution with 5 mg/ml PS concentration is introduced to miceintravenously, a dose is varied by the volume of introduced PS. Theresulting LD₅₀ value is 210.53±22.2 mg/kg, LD₁₀ value is 169.87 mg/kg.

EXAMPLE 15 In Vivo Biological Activity With the Use of the “Radachlorin,0.5% Solution for Injections” (“Photochlorin”) Medicinal Form

Photodynamic activity of the “Radachlorin, 0.5% solution for injections”(“Photochlorin”) medicinal form is studied on mice of the line Balb/cwith T36 embryocarcinoma inoculated into hind leg muscle. Mice weight is20-21 g. The irradiation procedure is realised with the diode laserML-662-SP 2 weeks after inoculation of a tumour. Skin in the irradiationarea is depilated before the procedure.

The preparation is introduced intraperotoneally in a dose of 40 mg/kg,that corresponds to a sufficient therapeutic dose. For carrying out theirradiation procedure the mice are etherised. The weight of tumours incontrol and experimental groups at the moment of experiment varies from0.9 up to 1 g. Irradiation is carried out 5-6 hours after injection ofPS. Every animal, except for control, is irradiated once, then it isobserved during a month after the procedure, the area of tumor necrosisand general physiological state are registered.

The average density of exposition dose of irradiation is 150 or 300J/cm².

The best results in the form of the complete tumour necrosis, crustformation in 1 week after PDT and this crust dropout in 1.5 months afterPDT are observed in the group that obtained the light dose of 300 J/cm².

EXAMPLE 16 Treatment of a Basal Cell Skin Cancer With the Use of the“Radachlorin, 0.5% Solution for Injections” (“Photochlorin”) MedicinalForm

The basal cell skin cancer is diagnosed at cytologic examination of ascrape. The preparation is introduced dropwise intravenously to make theconcentration of 0,7 mg/kg of patient's weight after dilution in 100 mlof 0,9% sterile NaCl saline solution. In 2-3 hours the irradiation of atumour with the diode laser ML-662-SP with wavelength of 662 nm with thesurface dose of 50 J/cm² is carried out without anaesthesia. Noundesirable side reactions are registered during injection of thepreparation and laser irradiation. In 2 hours after irradiation a darkbrown focus with surrounding redness zone of 1-2 cm is formed in theplace of the tumour. To the end of the first day a necrosis is formed inthe place of the tumour in the form of a dry dark brown crust (eschar).In 2-3 weeks the crust rejection takes place, and 2 weeks later the fullepithelisation of skin defect in the place of former basalioma takesplace with good cosmetic effect.

EXAMPLE 17 Treatment of a Basal Cell Skin Cancer With the Use of the“Radachlorin, 0.05% Gel” Medicinal Form

The basal cell skin cancer is diagnosed at cytologic examination of ascrape. The gel is applied over a tumour in thin layer, if possiblehealthy part of skin is not touched. The irradiation is carried out20-40 minutes after applying the gel. The irradiation procedure isrealised with the diode laser ML-662-SP (“Mylon-Sigm Plus”, Russia) withwavelength of 662 nm. The density of the exposition dose of irradiationis 2500 J/cm². In 2 hours after irradiation a dark brown focus withsurrounding redness zone of 1-2 cm is formed in the place of the tumour.In 1 week a necrosis is formed in the place of the tumour in the form ofa dry dark brown crust (eschar). In 2 weeks the crust rejection takesplace, and 2 weeks later the full epithelisation of skin defect in theplace of former basalioma takes place with good cosmetic effect.

EXAMPLE 18 Removing of a Tattoo With the Use of the “Radachlorin, 0.5%Solution in Dimethylsulfoxide for External Use” Medicinal Form

The solution is applied to the napkin and the latter is put over thetattoo, covered with black paper or thin aluminium foil and fixed for 30min. The excess of the solution is removed from a surface with cottonwetted with alcohol. The irradiation procedure is realised with thediode laser ML-662-SP (“Mylon-Sigm Plus”, Russia) with wavelength of 662nm, irradiation is being carried out along the pattern lines trying toavoid affecting the surrounding tissue. The density of the expositiondose of irradiation is 120 J/cm². In 1 hour after irradiation, skinredness and swelling is observed in the place of the tattoo. To the endof the second day a dark brown “picture” with surrounding redness zoneof 1 mm is formed. In 2 weeks a necrosis is formed in the place of thetattoo in the form of a dry dark brown crust. 2 weeks later the peelingof the crust together with the tattoo takes place. At smaller lightdoses the procedure goes without necrosis by stain decolorisation, butthere is a need in repeated sessions in this case. A soft pink tissue isformed in the place of the tattoo in 6 weeks as a result of PDT, thistissue slightly differing from the surrounding skin, the good cosmeticeffect is observed.

1-3. (canceled)
 4. A method for producing a photosensitizer according towhich Spirulina biomass is treated with acetone till chlorophyll a iscompletely extracted, the biomass is filtered out or centrifuged, theextract is treated with acid in order to remove magnesium ion out of thechlorophyll molecule and to hydrolyse phytyl ester group, and theresulting pheophorbide a derivative is reacted with a strong inorganicbase, characterized in that after treating the extract with acid forremoving magnesium ion out of the chlorophyll molecule the extract isneutralised and precipitated pheophytin a is filtered out, thenpheophytin a is hydrolysed in the mixture of hydrochloricacid-acetone-hexane, 6-16 ml acetone, 0.6-6-ml hexane and 5-10 mlconcentrated hydrochloric acid being used for every 1 g of crudepheophytin a, the mixture is heated up to 40-60° C. and stirred for 20min—1 hour, then hexane (6-16 ml) is added and organic phase is washedwith the mixture of acetone and hydrochloric acid (2-10:1), water phaseis washed with hexane, then water phase containing pheophorbide a isneutralised with excess of sodium citrate (tri-, di- ormono-substituted) water solution, precipitated pheophorbide a isfiltered out, washed with water, recrystallised out of the acetone—watermixture, air dried till its weight becomes constant, then pheophorbide ais dissolved in acetone, strong inorganic base is added in the form ofwater solution of 0.05-1.00% concentration, stirred at 30-60° C. for5-30 min, extra volume of strong inorganic base is added in the form ofwater solution of 1-50% concentration, the mixture is heated at 40-60°C. for 20-90 min, neutralised with diluted hydrochloric acid, chlorin e₆precipitate is separated by centrifugation, washed with distilled watertill acid reaction disappears, 55-80% of chlorin e₆ is obtained, thenchlorin e₆ is recrystallised out of acetone in order to separate lineartetrapyrroles, chlorin e₆ is filtered out and washed with distilledwater, chlorin e₆ is heated in sealed reservoir at the temperatures of40-100° C. for 1 hour—30 days, then it is cooled and strong basesolution is added till pH 7.5-8.5, then the solution is adjusted withapyrogenic water for injections to make photosensitizer concentration6.5-7.5% _(mass).
 5. A method for producing a photosensitizer of claim 4characterized in that after addition of the strong base solution till pH7.5-8.5 the mixture is gel filtrated to make chlorin e₆ percentage up to80-90%, purpurin 5—up to 5-20% and purpurin 18'the rest, then dilutedhydrochloric acid solution is added till photosensitizer precipitates,the solution is adjusted with apyrogenic water for injections to makephotosensitizer concentration 6.5-7.5% mass, so the “Liquid extract ofchlorins” is obtained.
 6. A method for producing a photosensitizer ofclaim 5 characterized in that after the stage of gel filtration dilutedhydrochloric acid solution is added to the photosensitizer solution tillphotosensitizer precipitates, then the precipitate is filtered out orseparated by centrifugation, the additives approved by RF StatePharmacopeia are added till pH 7.5-8.5, apyrogenic water for injectionsis added to make photosensitizer concentration 0.1-1% _(mass), thenbacteria are filtered out.
 7. A method for producing a photosensitizerof claim 5 characterized in that after the stage of gel filtrationdiluted hydrochloric acid solution is added to the mixture tillphotosensitizer precipitates, this precipitate is filtered out orseparated by centrifugation, adjusted with apyrogenic water forinjections to make photosensitizer concentration 6.5-7.5% _(mass), the“Liquid extract of chlorins” is dispersed in gel substrate according tohe following ratio: 0.5-12% _(mass) of the “Liquid extract of chlorins”,5-20% mas of dimethylsulfoxide, the rest is water, the pharmacologicallyacceptable additives and gel substrate.
 8. A method for producing aphotosensitizer of claim 5 characterized in that after the stage of gelfiltration diluted hydrochloric acid solution is added to the mixturetill photosensitizer precipitates, this precipitate is filtered out orseparated by centrifugation, adjusted with apyrogenic water forinjections to make photosensitizer concentration 6.5-7.5% _(mass), andthe resulting “Liquid extract of chlorins” is dissolved indimethylsulfoxide according to the following ratio: 0.5-12% _(mass) ofthe “Liquid extract of chlorins” and the rest is dimethylsulfoxide.